Light microscopy has been the essential technique for studying living cells since van Leeuwenhoek used his hand-made microscopes in the C17th. More recently, the development of laser-scanning confocal microscopy has revolutionized our understanding of many cellular functions by enabling the monitoring of cellular function in real time. Caged compounds (i.e. photosensitive, biologically inert signaling molecules) partner such optical techniques, as they provide control of cellular chemistry, in both temporal and spatial domains. One of the distinctive strengths of such exogenous chemical probes is that they active native membrane receptors upon photolysis. Thus, they nicely complement the recently developed channelrhodopsin technique. The goal of this proposal is the development and application of new chromophores designed for highly efficient uncaging of neurotransmitters. Synthetic organic chemistry will be used to make new chemical and optical neurobiology will be used to test the power of the probes for induction of synaptic plasticity in brain slices and living animals. During the past ten years a new type of solid-state laser technology has become readily available (Ti:sapphire), which allows for infra-red excitation of u.v.-absorbing chromophores. Uncaging using such lasers is produced by the simultaneous absorption of two red photons of equivalent energy to one blue photon. Since the excitation volume is approximately the same size as single synapse, 2-photon uncaging is uniquely suited to allow stimulation of selected synapses (one or many) on a dendritic surface. Since neurotransmitter release is created by light alone, the pattern of stimulation can be both arbitrary and rational. Specifically, we propose to make and test the following: (1) new caged glutamate probes that will allow the induction of synaptic plasticity in living animals; (2) new caging chromophores for dual-color, 2-photon uncaging, enabling spectrally independent, simultaneous photorelease of glutamate and GABA; (3) novel caging chromophores that are activated at longer wavelengths so enabling long-term optical stimulation experiments with reduced phototoxicity; (4) a diverse array of other caged neurotransmitters (e.g. AMPA, dopamine, adenosine, serotonin, agonists/antagonists of dopamine and serotonin receptors, etc.). The vast majority of excitatory synaptic transmission in the CNS occurs via glutamate receptors at spine heads. It is now well established that many neurological diseases (e.g. Alzheimer's, Down's, Huntingdon's, etc.) perturb spines in some way (size, number, distribution, etc.). Optical tools that enable the precise probing of synaptic function at the level of single spines are essential for a fuller understanding of the function of spines in normal and disease states. Our proposal is designed to fill part of this need. PUBLIC HEALTH RELEVANCE: The human brain is the most complex structure we know, having over one trillion synapses. Alzheimer's disease, Down's syndrome, and Huntingdon's disease are examples of extremely debilitating and deadly diseases that involve profound changes in the normal function of our brains. Such diseases target the smallest functional neuronal structures, namely individual synapses. Our work is designed to make probes that will allow us to study the precise function of one or many synapses in normal and disease states using modern laser technology.